Project Summary This project investigates how the tumor microenvironment (TME) impairs in situ interactions of T-cell surface molecules with counter-molecules on the melanoma cells to suppress anti-tumor immunity. Detailed mechanistic understanding will be obtained by an integrated approach that combines physical science (PS) based tools with two complementary pre-clinical mouse models of melanoma T cell immunity, which will be further tested using biospecimens from melanoma patients. The molecular focus is the T-cell receptor (TCR) that initiates the T-cell antigen recognition upon binding to peptide-major histocompatibility complex (pMHC), and the coreceptor CD8 that co-ligates with the pMHC. The first PS tool is quantifying TCR mechanosensing by single-molecule force probes through in situ kinetic analyses of molecular interactions with concurrent imaging of intracellular signals on a single cell. The second PS tool is DNA-based digital tension probes that report cell generated pulling forces on the TCR and CD8 via engaged pMHC. One animal model is a recognized standard that uses melanoma conjugated with a chicken ovalbumin antigen recognized by the OT-I TCR. The other animal model is a melanoma self-antigen gp100 in conjunction with JR209 humanized transgenic mice. By analyzing the mechanically regulated two-dimensional (2D) ligand binding of TCR and/or CD8 at the T-cell membrane, we observed that the TCR avidities for the pMHC of CD8 T cells infiltrating primary murine melanomas grown in vivo are significantly reduced relative to T cells within non-tumor associated tissues (spleen and blood). Such differential avidities were not detected by the conventional assay using pMHC tetramer, attesting to the power of our mechanics-based methods for analyzing TCR?pMHC interactions. We also found melanomas to substantially alter the force-dependent TCR?pMHC bond durability: in tumor-free animals, the TCR and pMHC formed a catch-slip bond whose lifetime first increased and then decreased with increasing force, which we have previously demonstrated to govern T cell signaling and effector function, whereas in melanoma-bearing animals, the TCR?pMHC bond lifetime only decreased with increasing force, i.e., behaved as a slip bond and were associated with reduced T cell effector functions. We hypothesize that deficient CD8 T cell immunity in melanoma results, at least in part, from impaired antigen recognition within the TME, as manifested by the altered TCR mechanosensing of pMHC. Three specific aims are proposed to test our hypothesis: 1) Determine the molecular interactions crucial to T cell antigen recognition that are impaired by the TME; 2) Define the functional consequences of suppressed T cell antigen recognition; and 3) Elucidate the mechanisms underlying the TME suppression of T cell antigen recognition. Completing these aims has the potential to identify new immunotherapeutic targets for the treatment of melanoma to improve the outcomes of patients with advanced disease.